In recent years, a study on a material for bio related research and bio medical service using magnetic substances has been frequently attempted. In particular, studies on a material for separating biomaterials and a use as a medical material have been actively attempted. Among others, a study on magnetic functional silane coated particles where organic functional compounds are coated on magnetic particles has been progressed so that the magnetic functional silane coated particles can be used for separation and purification of DNA and RNA, separation and purification of protein and amino acid, a bio sensor, a drug delivery system, magnetic resonance imaging (MRI) contrast agent, local warm-heating treatment, etc.
The magnetic particles start to be frequently applied as basic materials used in biotechnology research and have been used to quickly and simply separate the biomaterials. A method for separating biomaterials, in particular, nucleic acid or protein according to the related art needs much time and labor force since it should perform several extracting and centrifugal separating steps but degrades the yield and purity of the separated biomaterials and is not suitable for use as a method for automation or mass separation. However, in recent research, special magnetic particles were prepared and a method for very quickly and efficiently separating biomaterials using magnetic particles under appropriate buffer conditions was developed (U.S. Pat. No. 5,523,231, and U.S. Pat. No. 5,665,554).
Further, when using the above-mentioned method for separating biomaterials, an automation method, which can simultaneously process many samples and separate the biomaterials, can be provided. For example, when using a robot automatic apparatus, several hundreds or several thousands of samples can be automatically processed and desired biomaterials can be separated from the samples in large quantities.
In order to discriminate nucleic acid or protein from several cell mixtures, an effective and reproducible separating method is needed, and then a separating method using magnetic particles has been recently developed.
The separation of nucleic acid (DNA and RNA) from the biological samples is the most important step in a biochemistry research and diagnostic process.
If genetic materials (nucleic acid) are not separated from the samples, genetic detection, genetic cloning, genetic sequencing, genetic amplification, cDNA synthesis, and the like, which are subsequent steps, can not be performed. A method for separating nucleic acid using magnetic particles is a separating method that induces a bonding with biomaterials using the magnetic particles and then applies external magnetic field to samples, wherein it is known that the proper size of the magnetic particles used for separating and purifying DNA, RNA, protein, etc., is generally approximately 500 to 2000 nm.
As such, in order for the magnetic particles to be used for the separation and purification of gene (nucleic acid) or protein, it is preferable that they should have magnetic properties as well as should be conjugated with a functional group that bonds a gene or a specific protein on the surfaces of the particles. To this end, there is a need to coat the magnetic particles using the organic functional group or silica.
Among the magnetic particles used for separating the above-mentioned biomaterials, iron oxide particles are representative. The magnetic iron oxide particles generally exist as magnetite (Fe3O4), maghemite (Fe2O3), or hematite (Fe2O3) and the magnetic iron oxide particles can be used to separate and purify biomaterials, for example, nucleic acid (DNA and RNA), separate and purify protein, purify peptide and polypeptide, purify lipids, and the like.
The methods for preparing magnetic particles for separating biomaterials according to the related art can prepare the magnetic particles without agglomeration and interaction between the magnetic particles only by preparing magnetic iron or iron oxide particles from iron salt compounds using a liquid-phase reduction method and coating the prepared magnetic iron or iron oxide particles with silica, polymer or gold or silver, which are a non-magnetic materials. In recent years, among these magnetic particles, silica magnetic particles were being mainly developed (U.S. Pat. No. 6,027,945, U.S. Pat. No. 6,673,631, U.S. Pat. No. 7,183,002, and JP Patent No. 3253638). However, the silica magnetic particles has disadvantages in that a preparing process is complicated, the particle form is uneven, the separation yield in the separation and purification of biomaterials such as nucleic acid, and the like is degraded.
Meanwhile, a method for preparing silica magnetic particles having a spherical form from a method that forms a W/O type emulsion using sodium silicates aqueous solution and emulsifier, adds ammonium sulfate aqueous solution thereto, and then sufficiently agitates it is known in JP Laid-Open Patent No. 2001-136970 and 1994-047273. However, since the above method requires a process of dispersing the ammonium sulfate aqueous solution in the W/O type emulsion and a process of performing a sufficient agitation using an ultrasonic wave, etc., so that the dispersed ammonium sulfate aqueous solution micelle reacts with the sodium silicates aqueous solution micelle, the preparing process is complicated as well as since the size of the sodium silicates aqueous solution micelle is changed in the agitating process with the ammonium sulfate aqueous solution, it is difficult to uniformly control the silica particle size distribution to be formed.
KR Laid-Open Patent No. 2006-0061494 about the magnetic functional silica coated particles discloses a method for preparing magnetic functional silica coated particles by introducing an amine group or a chloro group on a surface as a method for preparing magnetic functional silica coated particles for separating and purifying nucleic acid, DNA, and RNA. However, the preparing method of the above-mentioned patent has disadvantages in that very expensive tetraethyl orthosilicate (TEOS) is used, the preparing process is complicated, and the particles are non-uniform. Further, KR Registration Patent No. 0541282 discloses a method that modifies the magnetic nanoparticles with silane materials and uses it; however, since the used magnetic nanoparticles themselves are magnetic substances, it has problems, such as biotoxicity, etc.